Protein-protein interaction of cytochrome b561 in chromaffin vesicle membranes studied by two-dimensional blue-native/sodium dodecyl sulfate gel electrophoresis and co-immunoprecipitation analysis
We analyzed a protein-protein interaction in solubilized chromaffin vesicles using two-dimensional electrophoresis (1st, Blue-Native PAGE; 2nd, Tricine-SDS-PAGE). Cytochrome b561 band, which was verified by immunoblotting, was observed in the two-dimensional gel with an apparent molecular weight of...
Elmentve itt :
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| Testületi szerző: | |
| Dokumentumtípus: | Cikk |
| Megjelent: |
2006
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| Sorozat: | Acta biologica Szegediensis
50 No. 1-2 |
| Kulcsszavak: | Természettudomány, Biológia |
| Online Access: | http://acta.bibl.u-szeged.hu/22776 |
| Tartalmi kivonat: | We analyzed a protein-protein interaction in solubilized chromaffin vesicles using two-dimensional electrophoresis (1st, Blue-Native PAGE; 2nd, Tricine-SDS-PAGE). Cytochrome b561 band, which was verified by immunoblotting, was observed in the two-dimensional gel with an apparent molecular weight of ~100~400kDa. On the other hand, purified cytochrome b561 showed a monomeric band (28 kDa) in Blue-Native PAGE. These results indicated that cytochrome b561 interacts with other proteins in the chromaffin vesicles to form a large protein complex(es). To clarify the nature of the interaction, we performed co-immunoprecipitation experiments, where the solubilized membrane proteins were treated with immunopurified anti- b561 IgG antibodies followed by sedimentation with protein-A-Sepharose. We found that there were no other proteins co-sedimented with cytochrome b561. Since the immunopurified anti- b561 IgG antibodies bound specifically to the C-terminal hydrophilic portion of cytochrome b561 protein, we concluded that such binding of the IgG antibodies to the C-terminal portion might cause an inhibition of protein-protein interaction with other proteins in the solubilized state. |
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| Terjedelem/Fizikai jellemzők: | 83-87 |
| ISSN: | 1588-385X |